Quantitative Assessment of Heat Shock-Induced Ferroptosis-Like Cell Death via Electrolyte Leakage in Arabidopsis thaliana Seedlings.

Abstract

We present a protocol to allow continuous assessment of cell death in Arabidopsis thaliana (L.) seedlings by measuring the release of electrolytes from dying cells upon heat shock. The electrolyte leakage assay is a well-established method to quantify the extent of cell death of plant tissues exposed to pathogen infection, since the activation of the immune response leads to compromised membrane integrity and to the release of ions from the dying cell. This prolonged release of electrolytes is considered a hallmark of regulated cell death in plants. Heat shock in plants induces ferroptosis-like cell death, which can be suppressed either pharmacologically, using inhibitors such as ferrostatin, or genetically through knockout of ferroptosis-related genes. Here, we have adapted the electrolyte leakage assay to quantify cell death in young Arabidopsis seedlings exposed to a heat shock previously shown to induce ferroptosis-like cell death. We also illustrate how this method can be used to assess activation of ferroptosis-like cell death in whole Arabidopsis seedlings using ferrostatin or knockout mutants of potential gene candidates involved in ferroptosis-like cell death. Key features • This protocol does not require any technical experience apart from gentle handling of young seedlings and is less labor-intensive than microscopy-based cell death evaluation. • Builds upon existing methods to quantify the extent of cell death upon immune response in whole seedlings subjected to heat stress. • Only requires a conductivity meter and allows the assessment of continuous cell death using multiple parallel replicates. • The protocol demonstrates how heat shock-induced ferroptosis-like cell death can be inhibited pharmacologically or genetically in whole seedlings, supported with quantitative data.

Plant phenotyping relevance

Arabidopsis幼植物の細胞死という植物状態を、電解質漏出で連続定量する測定プロトコルの適応・技術的提示が中心であり、ルーチン測定ではない。

Code and data availability

This is a Bio-protocol methods article describing an electrolyte leakage assay for heat shock-induced cell death in Arabidopsis seedlings. The supplied blocks contain no public phenotype/trait dataset, plant image or sensor data deposit, author analysis code, or trained model. Data analysis was done in GraphPad Prism,

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